Disk Susceptibility Testing Research Articles

Emergence of resistance to beta-lactam and aminoglycoside antibiotics during moxalactam therapy of Pseudomonas aeruginosa infections.

In four patients with Pseudomonas aeruginosa infections, the infecting strain developed resistance to moxalactam during therapy with this drug. In addition, P. aeruginosa isolates from two of these four patients showed increased resistance to aminoglycosides. Isolates from a third patient acquired cross-resistance to other antipseudomonal beta-lactams. In three of the cases, disk susceptibility tests failed to detect the resistance that was demonstrated in broth dilution assays. Isolate identities were confirmed by serotyping. No new plasmids were found by agarose gel electrophoresis. The mechanisms for this resistance did not involve enzymatic antibiotic degradation. These findings suggest that currently available expanded-spectrum cephalosporin derivatives should probably not be used alone for most serious infections due to P. aeruginosa. They also suggest that strains with multiple antibiotic resistance may become more prevalent in hospitals if these drugs are used extensively.

Cefoperazone: regression analysis, disk content, and disk susceptibility testing considerations.

Cefoperazone agar dilution minimal inhibitory concentration (MIC) susceptibility results were compared with zones of inhibition produced by disk diffusion susceptibility testing. Disks containing 30, 50, 75, and 100 micrograms of cefoperazone were tested for purposes of regression line comparisons and error rate-bounded analysis. Results suggest that if the MIC equivalent of susceptibility is 32 micrograms/ml, either a 50-micrograms disk with zone sizes of susceptibility (S) greater than or equal to 15 mm and resistance (R) less than or equal to 12 mm or a 75-micrograms disk with zone sizes of S greater than or equal to 17 mm and R less than or equal to 14 mm may be appropriate. If the MIC equivalent of susceptibility is 64 micrograms/ml, this would dictate use of a 75-micrograms disk with zone sizes of S greater than or equal to 17 mm and R less than or equal to 12 mm. Disks containing either less than or equal to 30 or greater than or equal to 100 micrograms of cefoperazone did not adequately discriminate among susceptible and resistant bacterial strains at either the 32- or 64-micrograms/ml MIC equivalent.

Treatment of infections due to methicillin-resistant Staphylococcus aureus.

Strains of methicillin-resistant Staphylococcus aureus are resistant to other penicillins. The in-vitro susceptibility to the cephalosporins differs among strains. Some strains, susceptible to cephalosporins by the standard disk susceptibility test, are proved resistant by the quantitative dilution test; they may show pop-up colonies within the zone of inhibition when incubated further at room temperature. The clinical efficacy of the cephalosporins with or without an aminoglycoside in treating infections due to methicillin-resistant S. aureus is in doubt. To date, susceptible to vancomycin. In-vitro antagonism of vancomycin and rifampin against S. aureus has been shown. Thus, vancomycin alone appears to be the treatment of choice; if this treatment fails, aminoglycoside or rifampin should be added. Serum bactericidal titers should be carefully monitored before and after the addition of the new agent and in-vitro time-kill studies of combination of antibiotics done if feasible.

Clinical impact of rapid antimicrobial susceptibility testing of blood culture isolates.

The results of a rapid, direct blood culture disk susceptibility test indicated that antimicrobial chemotherapy should be changed in 48 of 173 patients with bacteremia. In 32 patients (66.6%), the indicated change was made approximately 24 h sooner than if conventional, nonrapid susceptibility tests had been used to guide therapy.

Use of trimethoprim-sulfamethoxazole for treatment of infections due to Nocardia.

Trimethoprim-sulfamethoxazole (TMP-SMZ) was used for treatment of 34 patients with pulmonary or cutaneous nocardiosis. Of nine patients with primary cutaneous disease, eight had rapid resolution of their infection after short-term therapy and none have relapsed after a follow-up of more than six months. The 25 patients with pulmonary nocardiosis had a good clinical response, but three of five (60%) who completed less than three months of therapy relapsed within four weeks. Of the 10 patients who completed four to six months of therapy, only one (10%) relapsed and this relapse was due to drug resistance. By the method of serial dilution in agar, 96% of 59 isolates of Nocardia had MICs of SMZ of less than 25 micrograms/ml. Fewer than 20% were susceptible to 2.5 micrograms of TMP/ml. Synergy between TMP and SMZ was usually present with ratios of TMP to SMZ of 1:5, 1:1, or 5:1, but was less common at a ratio of 1:20. Disk susceptibility testing was easy to perform and readily separated sensitive from resistant strains. TMP-SMZ is highly effective for the treatment of nocardiosis, but the question of whether it is more effective clinically than a sulfonamide alone remains unanswered.

Agar disk diffusion for the quality control testing of Autobac elution disks.

The standard disk diffusion method of Bauer et al. (Am. J. Clin. Pathol. 45:493-496, 1966; National Committee for Clinical Laboratory Standards, Performance standards for antimicrobial disc susceptibility tests, approved standard ASM-2, 2nd ed., 1979) was used to monitor the potency of 16 Autobac antibiotic elution disks over an 8-month period. Results obtained by using the three recommended Autobac control strains showed excellent reproducibility. Zones sizes were all within a 5-mm range, and standard deviations ranged from 0.6 to 1.3 mm.

Evaluation of the rapid penicillinase paper strip test for detection of beta-lactamase.

The penicillin-starch paper strip method was compared with the acidometric and iodometric methods for assaying beta-lactamase production, using fresh isolates of clinically important bacteria. Results obtained by the three methods were compared for rapidity, accuracy, and stability of reagents. Of the 210 isolates tested by the paper strip method, 301 isolates tested by the acidometric method, and 117 isolates tested by the iodometric method, all were in perfect agreement with the disk diffusion susceptibility test except one strain each of Haemophilus influenzae, Staphylococcus aureus, and Staphylococcus epidermidis. The H. influenzae isolate was penicillin resistant and failed to give a positive test for beta-lactamase in all three tests. The staphylococci (intermediate and resistant in susceptibility, respectively) failed to give a positive test for beta-lactamase with the iodometric method. The results of the paper strip method, in which 3,241 strains representing nine species of bacteria were used, correlated completely with disk susceptibility tests except for 2 and 69 strains, respectively, of penicillin-resistant, beta-lactamase-negative H. influenzae and H. parainfluenzae. The results of this study indicate that the paper strip method is accurate, simple to perform, extremely economical, and uses materials that are stable when stored frozen. It is eminently suitable for routine laboratory use.

Strain identification inRhizobium meliloti using the antibiotic disk susceptibility test

The antibiotic disk susceptibility test was used to measure the variation in the intrinsic resistance of 49 strains ofRhizobium meliloti to 9 antibiotics. Several strains had unique patterns of resistance. However, during cluster analysis, when a minimum Euclidean distance equal to 4 was used as a discriminating tool, the strains were grouped in 12 groups. The largest group contained 74% of the strains but 9 strains (2 very effective, 4 effective and 3 ineffective) showed very unique patterns of resistance and formed 9 distinct groups.R. meliloti strains in general showed high intrinsic resistance to the 9 antibiotics tested.

Ceftizoxime: collaborative multiphased in-vitro evaluation including tentative interpretive standards for disc susceptibility tests, beta-lactamase stability, and inhibition.

Ceftizoxime: collaborative multiphased in-vitro evaluation including tentative interpretive standards for disc susceptibility tests, beta-lactamase stability, and inhibition.

Evaluation of a direct blood culture disk diffusion antimicrobial susceptibility test.

A total of 556 unique blood culture isolates of nonfastidious aerobic and facultatively anaerobic bacteria were examined by direct and standardized disk susceptibility test methods (4,234 antibiotic-organism comparisons). When discrepancies which could be accounted for by the variability inherent in disk diffusion susceptibility tests were excluded, the direct method demonstrated 96.8% overall agreement with the standardized method. A total of 1.6% minor, 1.5% major, and 0.1% very major discrepancies were noted.

Mezlocillin: tentative interpretive standards for disk diffusion susceptibility testing.

The susceptibility of 447 clinical bacterial isolates to mezlocillin and carbenicillin was tested by standardized agar disk diffusion and reference broth micro-dilution methods. Tentative interpretive criteria for disk susceptibility testing by using 75 micrograms mezlocillin disks are proposed: susceptible, greater than or equal to 16 mm; indeterminate, 13 to 15 mm; and resistant, less than or equal to 12 mm. These would be applicable to both Pseudomonas species and the Enterobacteriaceae, but not to Staphylococcus aureus. For S. aureus, the breakpoints for susceptible, greater than or equal to 29 mm, and resistant, less than or equal to 28 mm, hold for mezlocillin as well as for the other penicillinase-susceptible penicillins.

Erythromycin-Resistant Group A Streptococci-Reply

<i>In Reply</i>.—The results obtained by Dr Garber demonstrate very well how microorganisms of a single species are not static, but vary in their properties from place to place and from time to time. His bacteriological studies were performed by a technique identical to ours and in conformity with the accepted Kirby-Bauer method.¹Our laboratory consistently found a zero zone of inhibition to erythromycin on disk-susceptibility testing, with daily quality control to ensure disk antimicrobial potency. Because of expressed interest in the question of resistance to antibiotics, we are planning a follow-up study of streptococcal susceptibility to seek corroboration of our findings. Certainly the susceptibilities of group A β-hemolytic streptococci to antibiotics should be monitored. Because the justification for treatment in the individual patient is the prevention of rheumatic fever, we believe that follow-up cultures of throat material should be made when the patient has been treated with an

Gentamicin and amikacin disk susceptibility tests with Pseudomonas aeruginosa: definition of minimal inhibitory concentration correlates for susceptible and resistant categories.

With currently recommended disk susceptibility tests, minimal inhibitory concentration correlates for amikacin were greater than 16 micrograms/ml for resistance and less than or equal to 12 micrograms/ml (not less than or equal to 8 micrograms/ml) for susceptibility. For gentamicin, they were greater than 8 micrograms/ml for resistance and less than or equal to 6 micrograms/ml (not less than or equal to 4 micrograms/ml) for susceptibility. Minor discrepancies between disk tests and minimal inhibitory concentration determinations will occur if only doubling dilutions of drug are used for measuring minimal inhibitory concentration values.

Tentative interpretive standards for disk susceptibility tests with moxalactam (LY127935).

Moxalactam (LY127935; 6059-S) is a new beta-lactam antibiotic. We propose tentative zone standards for agar diffusion susceptibility tests with 30-microgram disks. The final selection of minimal inhibitory concentration breakpoints for definition of resistant and susceptible categories must await clinical experience with this drug. Some of the clinical questions to be answered are defined. A moderately susceptible (intermediate) category is proposed for those strains with minimal inhibitory concentrations of 16 or 32 microgram/ml (zones 15 to 22 mm in diameter). Strains with minimal inhibitory concentrations of greater than or equal to 64 microgram/ml are considered resistant, and those with minimal inhibitory concentrations of less than or equal to 8 microgram/ml are considered susceptible. Tests with 30-microgram disks did not satisfactorily separate strains with minimal inhibitory concentrations of 8 microgram/ml from strains requiring < 2 microgram/ml for inhibition, because the regression line became parabolic at concentrations of 2 microgram/ml and below. However, the disk tests were satisfactory for categorizing isolates into the above-described susceptible, moderately susceptible (intermediate), and resistant categories.

Interpretive standards for disk susceptibility tests with Sch 21420 and amikacin.

Disk susceptibility tests with two structurally related aminoglycosides (amikacin and Sch 21420) were evaluated. Tests with 10- and 30-micrograms amikacin disks confirmed previous recommendations for interpretive zone standards; 30-micrograms disks are preferred. Tests with 10-, 20-, and 30-micrograms Sch 21420 disks led to similar conclusions. The 30-micrograms Sch 21420 disks are recommended, with zone standards of less than or equal to 14 mm for the resistant category (minimal inhibitory concentration, greater than or equal to 32 micrograms/ml) and greater than or equal to 17 mm for the susceptible category (minimal inhibitory concentration, less than or equal to 16 micrograms/ml). If a minimal inhibitory concentration breakpoint of less than or equal to 8 micrograms/ml is preferred for defining the susceptible category, somewhat different zone standards may be used (less than or equal to 15 mm and greater than or equal to 19mm). Further evaluation documented the fact that tests with 30-micrograms amikacin disks predicted resistance or susceptibility to Sch 21420 almost as well as did a 30-micrograms Sch 21420 disk. Thus, the class concept of disk testing was judged to applicable, and routine testing with Sch 21420 may not be required.

Antibiotic resistance in Enterotoxigenic and non-enterotoxigenic Escherichia coli.

Antibiotic disk susceptibility tests were done on 220 strains of Escherichia coli belonging to serotypes reported in the literature to be associated with the production of enterotoxin. A total of 128 (58%) were resistant to one or more antibiotics, sulfa drugs, or chemotherapeutic agents. An analysis of these strains revealed primary, secondary, and tertiary drug resistance patterns that indicated a selective pattern in the formation of multiple drug resistance in E. coli. Resistances to certain antibiotics were more likely to occur in pairs and triads (secondary resistance patterns) that were often combined or coexisted in a single strain of E. coli to produce tertiary drug resistance patterns, conferring drug resistance to five or six different antibiotics. Among enterotoxin-associated serotypes, single and multiple drug resistance was less frequently associated with enterotoxin-produced strains than with strains from the same serotype that were not enterotoxigenic. Within the enterotoxigenic E. coli, single and multiple resistance to antibiotics was more frequent in strains producing only heat-stable enterotoxin (ST) than in strains producing only heat-labile enterotoxin (LT) or both. The number of resistances to different antibiotics per resistant strain averaged approximately 1.4 for LT plus ST or LT strains, and 3.9 for ST strains and nonenterotoxigenic strains. Phenotypic characterization of 170 strains for four usually plasmid-mediated characteristics showed that the number of antibiotics to which a strain was directly resistant varied with the type and number of plasmid-mediated characteristics present.

Cefotaxime: in vitro activity and tentative interpretive standards for disk susceptibility testing.

Tested against 9,412 recent clinical isolates, cefotaxime exhibited 8 to 64 times greater activity against the Enterobacteriaceae than did cephalothin and two to four times greater activity against Pseudomonas aeruginosa, but only one-half to one-eighth the activity of cephalothin against staphylococci. Using 420 different clinical isolates, but with comparable minimal inhibitory concentration (MIC) distributions, disk diffusion-MIC regression analyses were performed, using 5- and 30-micrograms cefotaxime disks. Cefotaxime MIC susceptible and resistant breakpoints of less than or equal to 8 and greater than 32 micrograms/ml are tentatively proposed. Based on the MIC breakpoints, the data showed the best discrimination among the three susceptibility categories (susceptible, indeterminate, and resistant) when the 30-micrograms cefotaxime disk was used. The zone diameter breakpoints as determined by the error rate-bounded method and regression analysis were greater than or equal to 23 mm for susceptible, 15 to 22 mm for indeterminate, and less than or equal to 14 mm for resistant.

Antagonism of cefamandole by cefoxitin in routine disk susceptibility tests.

Cefoxitin was found to antagonize cefamandole in standardized disk diffusion susceptibility tests. This antagonism increased as the distance between the two disks decreased, and was most frequently observed in tests with cephalothin-resistant Enterobacteriaceae. It occurred in tests with 68 of 98 (69%) cephalothin-resistant isolates, one of six (17%) cephalothin-intermediate isolates, and one of 40 (3%) cephalothin-susceptible isolates. Clinical laboratories that use both disks in routine susceptibility tests should be aware of this anatagonism and should ensure that the disks are not placed in proximity to each other.

Methods of measuring zones of inhibition with the Bauer-Kirby disk susceptibility test.

Standard Bauer-Kirby disk tests were performed with 85 selected isolates, each tested in triplicate by four different investigators. Each disk test was observed, and zone diameters were measured, under two lighting conditions (transmitted light and reflected light). The two lighting systems produced similar zone measurements (+/-2 mm) with 96% of the tests. When there were greater differences, zones appeared to be larger when observed with reflected light. Interlaboratory reproducibility was much greater when using reflected light rather than transmitted light. We concluded that zone diameters should be measured from the back of the plate while it is resting on, or held 2 to 3 inches [ca. 5.1 to 7.6 cm] above, a black, nonreflecting, flat surface, illuminated by a reflected light source.

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007